LOCUS       sequence-489179-        5325 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter J1-BBa_J23117-mRFP with expression of MS2 scRNA J106.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5325)
  AUTHORS   Kiattisewee C, Karanjia AV, Cardiff RAL, Olander KE, Leejareon P, 
            Alvi SS, Carothers JM, Zalatan JG
  TITLE     Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene
            Activation Reveals Antagonistic Effects.
  JOURNAL   ACS Synth Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358.
  PUBMED    40693287
REFERENCE   2  (bases 1 to 5325)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358."
COMMENT     Sequence Label: pCK161.06
FEATURES             Location/Qualifiers
     source          1..5325
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        148..182
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        266..284
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             307..336
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             352..369
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(425..442)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      595..641
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(694..711)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1212..1246
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     RBS             1256..1267
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1274..1951
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1984..2055
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2071..2098
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2629..3579)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     terminator      complement(4341..4435)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(4465..5256,5257..5325))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5088..5107
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gtggcaattc cgacgtcttg acagctagct cagtcctagg tataatacta
      181 gtaggacgcc tttggtaacc gcgttttaga gctagaaata gcaagttaaa ataaggctag
      241 tccgttatca acttgaaaaa gtggcacatg aggatcaccc atgtgctttt tttgaagctt
      301 gggcccgaac aaaaactcat ctcagaagag gatctgaata gcgccgtcga ccatcatcat
      361 catcatcatt gagtttaaac ggtctccagc ttggctgttt tggcggatga gagaagattt
      421 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg
      481 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
      541 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
      601 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
      661 tggatcctta ctcgagtcta gactgcaggc ttcctcgctc actgactcgc tgcgctcggt
      721 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
      781 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
      841 taaaaacgcc tacggtatcc accggtgcca cctgtggcaa ttccgacgtc gcctacggta
      901 tccacctgtg gcaattccga cgtcgcctac ggtatccacc ggagacctat ggcagccgaa
      961 aagtgccacc tgtggcaatt ccgacgtcgc ctacggtatc caccggagac gaaaagtgcc
     1021 acctgtggca attccgacgt cgcctacggt atccaccgga gacctatggc agcctccggc
     1081 cgccatagga cacctttggt tgccaagggt gacctatggt gaccatgggc caccacgggc
     1141 gacctcaggt atcctgcggt gtcctgcggt taccaaaggc gtcctttggg ttccaccgga
     1201 tacctccgga cttgacagct agctcagtcc tagggattgt gctagcgaat tcattaaaga
     1261 ggagaaaggt accatggcga gtagcgaaga cgttatcaaa gagttcatgc gtttcaaagt
     1321 tcgtatggaa ggttccgtta acggtcacga gttcgaaatc gaaggtgaag gtgaaggtcg
     1381 tccgtacgaa ggtacccaga ccgctaaact gaaagttacc aaaggtggtc cgctgccgtt
     1441 cgcttgggac atcctgtccc cgcagttcca gtacggttcc aaagcttacg ttaaacaccc
     1501 ggctgacatc ccggactacc tgaaactgtc cttcccggaa ggtttcaaat gggaacgtgt
     1561 tatgaacttc gaagacggtg gtgttgttac cgttacccag gactcctccc tgcaagacgg
     1621 tgagttcatc tacaaagtta aactgcgtgg taccaacttc ccgtccgacg gtccggttat
     1681 gcagaaaaaa accatgggtt gggaagcttc caccgaacgt atgtacccgg aagacggtgc
     1741 tctgaaaggt gaaatcaaaa tgcgtctgaa actgaaagac ggtggtcact acgacgctga
     1801 agttaaaacc acctacatgg ctaaaaaacc ggttcagctg ccgggtgctt acaaaaccga
     1861 catcaaactg gacatcacct cccacaacga agactacacc atcgttgaac agtacgaacg
     1921 tgctgaaggt cgtcactcca ccggtgctta aggatccaaa ctcgagtaag gatctccagg
     1981 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     2041 tcggtgaacg ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt
     2101 tatacctagg gtacgggttt tgctgcccgc aaacgggctg ttctggtgtt gctagtttgt
     2161 tatcagaatc gcagatccgg cttcagccgg tttgccggct gaaagcgcta tttcttccag
     2221 aattgccatg attttttccc cacgggaggc gtcactggct cccgtgttgt cggcagcttt
     2281 gattcgataa gcagcatcgc ctgtttcagg ctgtctatgt gtgactgttg agctgtaaca
     2341 agttgtctca ggtgttcaat ttcatgttct agttgctttg ttttactggt ttcacctgtt
     2401 ctattaggtg ttacatgctg ttcatctgtt acattgtcga tctgttcatg gtgaacagct
     2461 ttgaatgcac caaaaactcg taaaagctct gatgtatcta tcttttttac accgttttca
     2521 tctgtgcata tggacagttt tccctttgat atgtaacggt gaacagttgt tctacttttg
     2581 tttgttagtc ttgatgcttc actgatagat acaagagcca taagaacctc agatccttcc
     2641 gtatttagcc agtatgttct ctagtgtggt tcgttgtttt tgcgtgagcc atgagaacga
     2701 accattgaga tcatacttac tttgcatgtc actcaaaaat tttgcctcaa aactggtgag
     2761 ctgaattttt gcagttaaag catcgtgtag tgtttttctt agtccgttat gtaggtagga
     2821 atctgatgta atggttgttg gtattttgtc accattcatt tttatctggt tgttctcaag
     2881 ttcggttacg agatccattt gtctatctag ttcaacttgg aaaatcaacg tatcagtcgg
     2941 gcggcctcgc ttatcaacca ccaatttcat attgctgtaa gtgtttaaat ctttacttat
     3001 tggtttcaaa acccattggt taagcctttt aaactcatgg tagttatttt caagcattaa
     3061 catgaactta aattcatcaa ggctaatctc tatatttgcc ttgtgagttt tcttttgtgt
     3121 tagttctttt aataaccact cataaatcct catagagtat ttgttttcaa aagacttaac
     3181 atgttccaga ttatatttta tgaatttttt taactggaaa agataaggca atatctcttc
     3241 actaaaaact aattctaatt tttcgcttga gaacttggca tagtttgtcc actggaaaat
     3301 ctcaaagcct ttaaccaaag gattcctgat ttccacagtt ctcgtcatca gctctctggt
     3361 tgctttagct aatacaccat aagcattttc cctactgatg ttcatcatct gagcgtattg
     3421 gttataagtg aacgataccg tccgttcttt ccttgtaggg ttttcaatcg tggggttgag
     3481 tagtgccaca cagcataaaa ttagcttggt ttcatgctcc gttaagtcat agcgactaat
     3541 cgctagttca tttgctttga aaacaactaa ttcagacata catctcaatt ggtctaggtg
     3601 attttaatca ctataccaat tgagatgggc tagtcaatga taattactag tccttttccc
     3661 gggtgatctg ggtatctgta aattctgcta gacctttgct ggaaaacttg taaattctgc
     3721 tagaccctct gtaaattccg ctagaccttt gtgtgttttt tttgtttata ttcaagtggt
     3781 tataatttat agaataaaga aagaataaaa aaagataaaa agaatagatc ccagccctgt
     3841 gtataactca ctactttagt cagttccgca gtattacaaa aggatgtcgc aaacgctgtt
     3901 tgctcctcta caaaacagac cttaaaaccc taaaggctta agtagcaccc tcgcaagctc
     3961 gggcaaatcg ctgaatattc cttttgtctc cgaccatcag gcacctgagt cgctgtcttt
     4021 ttcgtgacat tcagttcgct gcgctcacgg ctctggcagt gaatgggggt aaatggcact
     4081 acaggcgcct tttatggatt catgcaagga aactacccat aatacaagaa aagcccgtca
     4141 cgggcttctc agggcgtttt atggcgggtc tgctatgtgg tgctatctga ctttttgctg
     4201 ttcagcagtt cctgccctct gattttccag tctgaccact tcggattatc ccgtgacagg
     4261 tcattcagac tggctaatgc acccagtaag gcagcggtat catcaacagg cttacccgtc
     4321 ttactgtccc tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt
     4381 ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc
     4441 gagctcgtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4501 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4561 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4621 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4681 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4741 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4801 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4861 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4921 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4981 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5041 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5101 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5161 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5221 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5281 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcat
//
